Monoclonal antibodies specific for human interleukin-5

ABSTRACT

Monoclonal antibodies which specifically bind to human interleukin-5 (IL-5) are described. The antibodies can be used in assays for IL-5, to purify IL-5, or to characterize IL-5.

This is a continuation of application Ser. No. 08/154,402, filed Nov.19, 1993, now abandoned.

FIELD OF THE INVENTION

The present invention is in the field of recombinant DNA technology.This invention is directed to antibodies, and in particular, monoclonalantibodies that are capable of specifically binding to humaninterleukin-5. The invention concerns the development of suchantibodies, as well as their diagnostic and therapeutic uses.

BACKGROUND OF THE INVENTION

I. Interleukin-5 and the Humoral Response to Microbial Infection

Antibodies are produced by the immune system of humans and animals inresponse to the presence of molecules that are considered "foreign."Such molecules fall into two classes: "antigens" or "haptens." Anantigen is a molecule whose presence in an animal is capable of inducingthe immune system to produce antibodies. In contrast, a hapten iscapable of being bound by an antibody, but is not capable of elicitingantibody formation. Haptens are generally small molecules; whenconjugated to a larger molecule, they can become antigens, and thusinduce antibody formation. The nature and structure of antibodies, andthe tenets of immunology are disclosed by Davis, B. D. et al. (In:Microbiology, 2nd Ed., Harper & Row, NY (1973)).

The capacity to induce antibody formation in response to microbialinfection depends upon a series of interactions among T cells, B cells,and macrophages. Each B cell is genetically programmed to produce cellsthat express an immunoglobulin (or antibody) that is capable of specificinteraction with a distinct antigenic determinant on the infectiousmicrobe. Such antibodies play a central role in the humoral response toinfection (Takatsu, K., Microbial Rev 35:593-606 (1991)).

The B cell response to an antigen is regulated by a helper T cellresponding to, and specific for, the same antigen molecule (Takatsu, K.,Microbial Rev 35:593-606 (1991)). The helper T cells recognize antigenicpeptides in the context of class II major histocompatability complex(MHC) molecules that are arrayed on B cells, and secrete several solublefactors ("lymphokines") which can induce growth and maturation of Bcells (Howard, M. et al., Ann. Rev. Immunol. 1:307-333 (1983);Kishimoto, T. et al., Ann. Rev. Immunol. 3:133-157 (1985); Melchers, F.et al., Ann Rev. Immunol. 4:13-36 (1986); Takatsu, K., Microbial Rev35:593-606 (1991)).

One of these soluble factors (termed, "T-cell-replacing factor" ("TRF")was found to induce the terminal differentiation of activated B cellsinto antibody producing cells (Takatsu, K. et al., J. Immunol.124:2414-2422 (1980)). This factor was subsequently found to have anactivity (termed "BCGF II") that could promote DNA synthesis in certainB cell leukemic cells (Harada, N. et al., J. Immunol. 134:3944-3951(1985)). Further research revealed the additional presence of aneosinophil differentiation factor capable of inducing eosinophil colonyformation (Sanderson, C. J. et al., J. Exper. Med. 162:60-74 (1985)).Ultimately, these factors were purified (Sanderson, C. J. et al., Proc.Natl. Acad. Sci. (U.S.A.) 83:437-440 (1986)) and sequenced (Campbell, H.D. et al., Eur. J. Biochem, 174:345-352 (1988); Kinashi, T. et al.,Nature 324:70-73 (1986)), and found to be derived from a single protein,termed "interleukin-5" ("IL-5") (McKenzie, A. N. N. et al., In:Interleukins: Molecular Biology and Immunology, Chem. Immunol., Basel,Karger, vol. 51, pp 135-152 (1992), herein incorporated by reference).

The cDNA encoding murine IL-5-TRF/BCGFII was cloned by using SP6expression vector system (Kinashi, T., et al., Nature 324:70-73 (1986)).The amino acid sequence deduced from the nucleotide sequence of theentire IL-5 cDNA revealed that murine IL-5 consists of 133 amino acidsincluding a hydrophobic signal sequence of 20 amino acids (Kinashi, T.,et al., Nature 324:70-73 (1986)) with a molecular mass of 12.3 kDa.Thus, the mature human IL-5 protein has 113 amino acids, and is the samelength as the mature murine IL-5. Comparison of the cDNA sequence ofmurine IL-5 with that of human shows a sequence homology of 77% at theDNA level and 70% at the protein level.

Interleukin-5 is a homodimeric glycoprotein. Investigations usingreduced and alkylated IL-5 suggest that dimerization is essential forbiological activity (Tsuroka, N., et al., Cell. Immunol. 125:354-362(1990)). Comparison of the two polypeptide sequences shows 81 identicalamino acids, 25 conservative amino acid changes, with the remaining 7(and the two N-terminal amino acids of the human protein sequence) asnon-conservative changes. This high degree of similarity is reflected inthe ability of the two proteins to cross-react with cells of othermammals (Sanderson, C. J., et al., In: Colony Stimulating Factors:Molecular and Cellular Biology, Marcel Dekker, NY, pp 231-256 (1990)).Despite their capacity to cross react, murine and human IL-5 displaysignificant species specificity, with the murine material beingapproximately 100-fold more active against murine cells than the humanmolecule. Conversely, human IL-5 is approximately 20-fold more active inhuman bone marrow assays than murine IL-5)McKenzie, A. N. J. et al., In:Interleukins: Molecular Biology and Immunology, Chem. Immunol., Basel,Karger, vol. 51, pp 135-152 (1992)).

Native IL-5 or recombinant IL-5 expressed in mammalian cells inheterogeneously glycosylated, with the mouse sequence containing threepotential N-glycosylation sites, while the human sequence lacks on ofthese. Carbohydrate has been shown to be unnecessary for biologicalactivity, although it is possible that it may perform some role ingoverning the half-life of the polypeptide in the circulation (McKenzie,A. N. J. et al., In: Interleukins: Molecular Biology and Immunology,Chem. Immunol., Basel, Karger, vol. 51, pp 135-152 (1992)).

Interleukin-5 promotes the growth of B-lineage cells. IL-5 acts onnaturally activated B cells, on LPS-stimulated B cells, and on resting Bcells to induce maturation and to propagate proliferation (Karasuyama,H. et al., J. Exper. Med. 167:1377-1390 (1988)). IL-5 induces theincrease in levels of secreted forms of μ-mRNA in BCL₁ or resting aswell as activated B cells (Webb, C. P. et al. J. Immunol. 143:3934-3939(1989)). Murine IL-5 was found to cause an increase in the frequency ofB cells both proliferating and lg secreting (Alderson, M. R. et al., J.Immunol. 139:2656-2660 (1987)). IL-5 can induce antigen-specific andpolyclonal IgA production in antigen-primed B cells and inLPS-stimulating B cells, respectively.

IL-5 also appears to promote immunoglobulin formation. Transgenic micecarrying the IL-5 gene exhibited elevated levels of IL-5 in the serum(2-10 ng/ml), and an increase in the levels of serum IgM and IgA. Amarked increase in the number of peripheral blood white cells (PBL), ofspleen cells and of peritoneal cells was also observed. Particularly,the increase in the numbers of eosinophils in PBL reached 70-fold thoseof age-matched control mice (Tominaga, A. et al., J. Exper. Med.144:1345-1352 (1990)). Antibodies to IL-5 mAbs have been isolated(Coffman, R. L. et al., Science 245:308-311 (1989)), and have been foundto inhibit the antigent-specific primary IgM response induced by acloned helper T cells in an MHC-restricted manner and also to inhibitpolyclonal Ig-secretion (Rasmussen, R. et al., J. Immunol. 140:705-712(1988)).

Importantly, IL-5 also plays a role in the production and maturation ofeosinophils. Eosinophils are immune system cells that accumulate inresponse to allergic inflammatory reactions. They ingestantibody-antigen complexes and thereby become degranulated. Since thegranules contain substances capable of blocking the action of histamine,serotonin and bradykinin, all of which are involved in inflammation, ithas been proposed that eosinophils protect the tissues of the host notonly by phagocytizing and degrading cytotoxic antibody-antigen complexesbut also by damping the effects of chemical mediators of theinflammatory response (Davis, B. D. et al., In: Microbiology, 2nd Edit.,Harper & Row, NY (1973)). IL-5 thus plays an important part in diseasesassociated with increased iosiniphils, such as asthma and similarinflammatory conditions (Sanderson, C. J., In: Advances in Pharmacology,vol. 23, Academic Press, NY, pp. 163-177 (1992); Takatsu, K., Curr.Opin. Immunol. 4:299-306 (1992), both herein incorporated by reference).

Murine IL-5 has also been shown to induce the production of eosinophilsin liquid bone marrow cultures (McKenzie, A. N. J. et al., In:Interleukins: Molecular Biology and Immunology, Chem. Immunol., Basel,Karger, vol. 51, pp 135-152 (1992)). maintain the viability of matureeosinophils, to induce the production of superoxide anion in matureeosinophils and to possess chemotactic activity for eosinophils (Harada,N. et al., J. Immunol. 134:3944-3951 (1985); Lopez, A. F. et al., J.Exp. Med. 167:219-224 (1987); Owen, W. F. et al., J. Exper. Med.170:343-349 (1989); Yamaguchi, N. et al., J. Exper. Med. 167:43-56(1988); Yamaguchi, N. et al., J. Exper. Med. 167:1737-1742 (1988)). Thesyngergistic effect of IL-5 and colony-stimulating factors on theexpansion of eosinophils is supposed to contribute to the urgentmobilization of eosinophils at the time of helminthic infections andallergic responses.

Thus, IL-5 appears to play a significant role in inducing inflammatoryprocesses. Molecules that potentiate this activity are therefore highlydesired anti-inflammatory agents.

II. Antibodies and Immunoassays

Immunoassays are assay systems that exploit the ability of an antibodyto specifically recognize and bind to a particular target molecule.Immunoassays are used extensively in modern diagnostics (Fackrell, J.Clin. Immunoassay 8:213-219 (1985)). A large number of differentimmunoassay formats have been described (Yolken, R. H., Rev. Infect.Dis. 4:35 (1982); Collins, W. P., In: Alternative Immunoassays, JohnWiley & Sons, NY (1985); Ngo, T. T. et al., In: Enzyme MediatedImmunoassay, Plenum Press, NY (1985)).

The simplest immunoassay involves merely incubating an antibody that iscapable of binding to a predetermined target molecule with a samplesuspected to contain the target molecule. The presence of the targetmolecule is determined by the presence, and proportional to theconcentration, of any antibody bound to the target molecule. In order tofacilitate the separation of target-bound antibody from the unboundantibody initially present, a solid phase is typically employed. Thus,for example the sample can be passively bound to a solid support, and,after incubation with the antibody, the support can be washed to removeany unbound antibody.

In more sophisticated immunoassays, the concentration of the targetmolecule is determined by binding the antibody to a support, and thenpermitting the support to be in contact with a sample suspected tocontain the target molecule. Target molecules that have become bound tothe immobilized antibody can be detected in any of a variety of ways.For example, the support can be incubated in the presence of a labelled,second antibody that is capable of binding to a second epitope of thetarget molecule. Immobilization of the labelled antibody on the supportthus requires the presence of the target, and is proportional to theconcentration of the target in the sample. In an alternative assay, thetarget is incubated with the sample and with a known amount of labelledtarget. The presence of any target molecules in the sample competes withthe labelled target molecules for antibody binding sites. Thus, theamount of labelled target molecules that are able to bind the antibodyis inversely proportional to the concentration of target molecule in thesample.

In general, immunoassay formats employ either radioactive labels("RIAs") or enzyme labels ("ELISAs"). RIAs have the advantages ofsimplicity, sensitivity, and ease of use. Radioactive labels are ofrelatively small atomic dimension, and do not normally affect reactionkinetics. Such assays suffer, however, from the disadvantages that, dueto radioisotopic decay, the reagents have a short shelf-life, requirespecial handling and disposal, and entail the use of complex andexpensive analytical equipment. RIAs are described in LaboratoryTechniques and Biochemistry in Molecular Biology, by Work, T. S., etal., North Holland Publishing Company, NY (1978), with particularreference to the chapter entitled "An Introduction to Radioimmune Assayand Related Techniques" by Chard, T., incorporated by reference herein.

ELISAs have the advantage that they can be conducted using inexpensiveequipment, and with a myriad of different enzymes, such that a largenumber of detection strategies--colorimetric, pH, gas evolution,etc.--can be used to quantitate the assay. In addition, the enzymereagents have relatively long shelf-lives, and lack the risk ofradiation contamination that attends to RIA use. ELISAs are described inELISA and Other Solid Phase Immunoassays (Kemeny, D. M. et al., Eds.),John Wiley & Sons, NY (1988), incorporated by reference herein.

Antibodies that would be capable of specific binding to Interleukin-5would be highly desired aids in detecting or quantifying IL-5 presenceor levels. Although antibodies to the murine IL-5 have been identifiedthat also bind human IL-5, it would be desirable to have antibodies ofgreater specificity and/or avidity to human IL-5. Such reagents wouldalso be valuable in determining IL-5 derivatives and mimetics havingincreased or modified biological activity. The present inventionprovides such antibodies, as well as methods for their exploitation.

SUMMARY OF THE INVENTION

The present invention concerns antibodies, and in particular, monoclonalantibodies that are capable of specifically binding to humaninterleukin-5. The invention provides methods for forming suchmolecules, and for using such molecules to measure IL-5 levels in asample, as well as to design potential IL-5 therapeutics. Although theinvention particularly concerns murine monoclonal antibodies, it is alsodirected to the development of IL-5 ligands, and to humanizedantibodies.

In detail, the invention concerns an antibody, or fragment thereof,capable of specifically binding human interleukin-5, wherein theantibody or protein is substantially incapable of binding murineinterleukin-5.

The invention also concerns a cell (mammalian, insect, bacterial, etc.)capable of producing an antibody, or fragment thereof, that is capableof specifically binding human interleukin-5, wherein the antibody orprotein is substantially incapable of binding murine interleukin-5.

The invention also concerns a baculovirus vector capable of directingthe expression of human IL-5 in an insect cell, as well as the insectcell that contains such vector.

The invention provides a method for determining whether a candidateagent is capable of modulating a biological activity of humaninterleukin-5, which comprises incubating the candidate agent in thepresence of an antibody, or fragment thereof, capable of specificallybinding human interleukin-5, but substantially incapable of bindingmurine interleukin-5, and determining whether the agent is capable ofbeing bound by the antibody or fragment.

The invention further provides a method for determining the presence orconcentration of interleukin-5 in a sample which comprises contactingthe sample with an antibody, or fragment thereof, capable ofspecifically binding human interleukin-5, but substantially incapable ofbinding murine interleukin-5, and determining whether the samplecontains a molecule capable of binding to the antibody or fragment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graph of the neutralization of IL-5 bioactivity in theBCL1 proliferation assay. Each mAb was incubated with either 1 μg/mlrhIL-5 (solid bar) or 1 ng/ml rmIL-5 (stippled bar). Each numberindicates the respective anti-hIL-5 mAb, T=TRFK-5 (a positive controlmAb for neutralization), OK=media (no mAb). Samples were assayed intriplicate and data are expressed as mean ±SE.

FIG. 2 shows the dose dependency of mAb neutralization of rhIL-5. IL5.7,a group A mAb representative (open square), IL5.15, a group B mAbrepresentative (open triangle), TRFK-5, a positive control mAb forneutralization (open diamond), and anti-OKT11, an irrelevant isotypematched mAb (open circle) were titrated in serial two fold dilutionsstarting with 40 μg/ml and incubated with 1 μg/ms rhIL-5. All other mAbnot shown yielded similar results. Samples were assayed in triplicateand data are expressed as the mean ±SE.

FIGS. 3A-3B show western analysis of reduced and nonreduced rhIL-5. FIG.3A: Nonreduced rhIL-5 was electrophoresed and transferred to PVDFmembrane. Each number represent the respective anti-hIL-5 primary mAbincubated at 100 μg/ml. The (-) lane was run without primary mAb. FIG.3B: Conditions were the same as above except rhIL-5 was run underreducing conditions in all lanes except (+) which was a positive controlfor Western development and contained nonreduced rhIL-5 detected withIL5.5.

FIG. 4 shows the results of a human IL-5 sandwich ELISA. Plates werecoated with either IL5.7, IL5.15 or TRFK-5 (5 μg/ml). Serial two folddilutions of rhIL-5 starting with 50 ng/ml or 2 μg/ml were incubated inblocked wells. Biotinylated mAb, b-IL5.7, b-IL5.15, or b-TRFK-5 wereused for detection. Samples were assayed in triplicate with dataexpressed as the mean ±SE.

FIG. 5 shows the sensitivity of the hIL-5 sandwich ELISA. Plates werecoated with IL5.15 and detected with b-IL5.7. Dilutions of rhIL-5between 90 and 15 pg/ml were assayed in triplicate. Data are expressedas the mean of three experiments ±SE. Significance between points asestablished using the Tukey test (P<0.01).

DETAILED DESCRIPTION OF THE INVENTION

The potential role of IL-5 in the pathogenesis of inflammatory disorderssuggests that the interaction of IL-5 with its receptor can be targetedby IL-5 derivatives, and by IL-5 mimetic agents in order to modulatethis response. The present invention provides antibodies, and mostpreferably murine antibodies that are capable of specific binding tohuman IL-5 ("hIL-5"). As discussed below, such antibodies can be used inimmunoassay formats to assay IL-5 expression. They also permit theidentification of domains and/or residues of IL-5 that are functionallyrelevant to the biological activity of the molecule.

I. Interleukin-5

Interleukin-5 (IL-5) is a homodimeric cytokine secreted predominantly byactivated Th2 lymphocytes (Clutterback, E., et al., Eur. J. Immunol.17:1743 (1987); Takatsu, K., et al., Immun. Rev. 102:107 (1988);Enokihara, H., et al., Blood 73:1809 (1989), Parronchi, P., et al., Eur.J. Immunol. 22:1615 (1992); Schrezenmeier, et al., Int. Soc. Exp. Hemat.21:358 (1993)). The homodimer is covalently linked by two disulfidebonds in a head-to-tail configuration (McKenzie, A. H. J., et al.,Molecular Immunology 3:155 (1991); Tsuruoka N., et al., Cell. Immun.125:354 (1990); Minamitake, Y., et al., J. Biochem. 107:292 (1990)).Dimer formation is absolutely essential for biological activity asmonomeric IL-5 does not bind its receptor (McKenzie, A. H. J., et al.,Molecular Immunology 3:155 (1991); Tsuruoka N., et al., Cell. Immun.125:354 (1990); Takahashi T., et al., Molecular Immunology 27:911(1990); Tominaga, A., et al., J. Immunology 144:1345 (1990)). IL-5 isheterogeneously gylcosylated with both N-and O-linked residues. Althoughthese residues' may provide some thermal stability (Tominaga, A., etal., J. Immunology 144:1345 (1990)), they are not required forbiological activity (Kodama, S., et al., Eur. J. Biochim. 211:903(1993); Proudfoot, A. E. I., et al., Biochem. J. 270:357 (1990)). MurineIL-5 (mIL-5) and human IL-5 (hIL-5) share 73 percent amino acid homology(McKenzie, A. N. J., et al., EMBO 10:1193 (1991)), which enablescross-species reactivity in various bioassays. However, while human IL-5("hIL-5") and murine IL-5 ("mIL-5") act equally well in human cellassays, mIL-5 is greater than 100 fold more potent in murine cell assays(Plaetinch, G., et al., J. Exp. Med. 172:683 (1990)). This difference inpotency provides an excellent system in which to study species specificIL-5 domains. Using murine/human chimeric constructs of IL-5, speciesspecific activity has been localized to the C-terminal 36 amino acidresidues (McKenzie, A. N. J., et al., EMBO 10:1193 (1991)).

Recently the crystal structure of hIL-5 was solved revealing twofour-a-helix bundle motifs situated about a two fold axis of symmetry(Milburn, M. V., et al., Nature 363:172 (1993)). Although a four-a-helixbundle motif is characteristic for many cytokines, the IL-5 motif isunique in that for each bundle the three N-terminal helices of a givenchain are complemented by the C-terminal helix from the other chain.

Murine IL-5 has been characterized for its effects on growth,differentiation and activation of both B-cells and eosinophils (Kyoshi,T., Microbiol. Immunol. 35:593 (1991)). It has also been demonstrated toinduce differentiation of thymocytes to cytolytic T-cells and enhancetheir killing activity (Takatsu, K., et al., Proc. Natl. Acad. Sci.(U.S.A.) 84:4232 (1987); Aoki, T., et al., J. Exp. Med. 170:583 (1989)).Although the effect of hIL-5 on B-cells is controversial (Asuma, C., etal., Nucleic Acid Research 14:9146 (1986); Yokata, T., et al., Proc.Natl. Acad. Sci. U.S.A. 84:7388 (1987); Clutterbuck, E., et al., Eur. J.Immunol. 17:1743 (1987)). its effects on other hematopoietic cells isbetter established. Best characterized is the effects of hIL-5 oneosinophils. Human IL-5 selectively induces eosinophil differentiationand proliferation (Enokihara, H., et al., Blood 73:1808 (1989);Hirohisa, S., et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:2288 (1988)),increases eosinophil survival (Yamagushi, Y., et al., Blood 78:2542(1991); Wallen, N., et al., J. Immunol. 147:3490 (1991); Stern, M., etal., J. Immunol. 148:3543 (1992)), and acts as an iosinophil chemotacticfactor (Coeffier, E., et al., J. Immunol. 147:2595 (1991); Wang, J. M.,et al., Eur. J. Immunol. 19:701 (1989)). Eosinophil effector functionsare enhanced by hIL-5 as well (Coeffier, E., et al., J. Immunol.147:2595 (1991); Wang, J. M., et al., Eur. J. Immunol. 19:701 (1989);Fujisawa, T., et al., J. Immunol. 144:642 (1990)). IL-5 can enhancedifferentiation and activation of basophils, including the release ofhistamine and leukotrienes (Denburg, J. A., et al., Blood 77:2472(1991); Hirai, K., et al., J. Exp. Med. 172:1525 (1990); Bischoff, S.C., et al., J. Exp. Med. 172:1577 (1990)). These many effects haveimplicated hIL-5 in the pathogenesis of asthma, allergy, andhypereosinophilic disorders (Robinson, D. S., et al., N. Engl. J. Med.326:298 (1992); Hamid, Q., et al., J. Clin. Invest. 87:1541 (1991);Corrigan, C. J., et al., Am Rev. Respir. Dis. 147:540 (1993); Durham,S., et al., J. Immunol. 148:2390 (1992); Sehmi, R., et al. Blood 79:2952(1992); Ohnishi, T., et al., Am. Rev. Respir. Dis. 147-901 (1993)).

II. The Production of Monoclonal Antibodies against Interleukin-5

Any of a variety of methods can be used to permit the production ofmonoclonal antibodies that are capable of specific binding to IL-5. Inone embodiment, IL-5 can be purified from natural sources, in order toobtain samples sufficient to immunize an animal. Most preferably, theIL-5 immunogen will be produced via recombinant means through thecloning and expression of recombinant hIL-5 ("rhIL-5"). One aspect ofthe present invention concerns the capacity to produce high levels ofsubstantially purified rhIL-5. As used herein, the term "substantiallypurified" rhIL-5 is intended to describe a rhIL-5 preparation which issubstantially or completely lacks one or more portein, lipid, orcarbohydrate that is normally associated with, or present in crudepreparations of human IL-5.

A. The Cloning of Human IL-5 cDNA

Most preferably, such cloning is accomplished by adapting a hIL-5 cDNAsequence into a vector capable of expression in a baculoviral expressionsystem (see, Summers, M. D., et a., A Manual Of Methods For BaculovirusVector And Insect Cell Culture Procedures, The Texas AgriculturalExperiment Station, College Station, Tex. (1987)). A preferred source ofhIL-5 cDNA is plasmid phIL5 115.1, which is obtainable from the AmericanType Culture Collection, Rockville, Md., U.S.A. as accession number ATCC59394. The original cDNA was isolated and cloned into pcDVI vector byYokata, T., et al., Proc. Natl. Acad. Sci. U.S.A. 84:7388 (1987)) usinga homopolymeric tailing method. This insert including G-C tails wasexcised from its original vector and inserted in to the BamH1 side ofPBR 322 by the ATCC.

It is desirable to modify the ATCC 39394 vector in order to maximizeexpression. Specifically, high levels of expression can be obtained bymodifying the plasmid such that the normal hIL-5 start codon becomes outof frame and downstream of the mutated polyhedron start codon, ATT. Themodified vector is preferably co-transfected with wild type AcMNPV, orits equivalent, into a suitable cell line, such as the Sf9 cell line,and recombinant viruses picked and subcloned by plaque assay andhybridization using standard methodology (Summers, M.D., et al., AManual Of Methods For Baculovirus Vector And Insect Cell CultureProcedures, The Texas Agricultural Experiment Station, College Station,Tex. (1987)).

Recombinant hIL-5 (rhIL-5) was preferably produced in spinner flasks ofSf9 cells using a multiplicity of infection of approximately ten.Preferably, the cell culture is filtered after several days ofinfection, and the supernatant is recovered, treated with detergents,and passed over a CnBr-Sepharose column that has been derivatized withthe antibody to murine IL-5. The preferred murine antibody for thispurpose is TRFK-5 (Mita, S., et al., J. of Immunol. Meth. 125:233(1987)). Most preferably, the column purification of rhIL-5 is conductedas described by Schumacher, J. H., et al. (J. Immuno. 141-1576 )(1988)). The purity of the recovered rhIL-5 can be tested by silverstaining following SDS-PAGE and can be quantiated using the microliterBCA protein assay (Pierce, Rockford, Ill.) and by ELISA using TRFK-5 ina double antibody sandwich as described (Mita, S., et al., J. ofImmunol. Meth. 125:233 (1987)). Functional quantitation can be doneusing the murine BCL, proliferation assay (Mita, S., et al., J. Immunol.Meth. 125:233 (1987)).

B. The Induction of Monoclonal Antibodies

Monoclonal antibodies that are capable of specific binding to hIL-5 arepreferably produced by immunizing mice with the above-describedaffinity-purified rhIL-5.

BALB/c mice are preferred for this purpose, however, equivalent strainsmay also be used. The animals are preferably immunized withapproximately 25 μg of affinity purified baculovirus derived rhIL-5 thathas been emmusified 1:1 in TiterMax adjuvant (Vaxcel, Norcross, Ga.).Immunization is preferably conducted at two intramuscular sites, oneintraperitoneal site, and one subcutaneous site at the base of the tail.An additional i.v. injection of approximately 25 μg rhIL-5 is preferablygiven in normal saline three weeks later. After approximately 11 daysfollowing the second injection, the mice may be bled and the bloodscreened for the presence of anti-hIL-5 antibodies. Preferably, a directbinding ELISA is employed for this purpose.

Most preferably, the mouse having the highest anti-hIL-5 titer is givena third i.v. injection of approximately 25 μg rhIL-5. The splenicleukocytes from this animal may be recovered 3 days later, and are thenpermitted to fuse, most preferably, using polyethylene glycol,. withcells of a suitable myeloma cell line. A preferred myeloma cell line isthe p3X63Ag8.653 myeloma cell line. Hybridoma cells are selected byculturing the cells under "HAT" (hypoxanthine-aminopterin-Thymine)selection for about one week. The resulting clones may then be screenedfor their capacity to produce monoclonal antibodies ("mAbs") to hIL-5("anti-hIL-5"), preferably by direct ELISA.

High level production of the anti-hIL-5 mAbs was obtained using nudemice. Nude mice are primed with 0.5 ml of2,6,10,14-tetramethypentadecane (Aldrich, Milwaukee, Wis.). Afterapproximately 5 days, each clone is harvested, pelleted, and resuspendedin sterile PBS to a final destiny of approximately 2.5×10⁶ cell/ml. Apair of nude mice were injected for each monoclonal antibody. Antibodymay be recovered from the ascites fluid of the animals, and ispreferably lipocleaned with Seroclear (Calbiochem, San Diego, Calif.)following vender specifications. The mAbs may then be further purified,preferably using a GamaBind Plus Sepharose column (Pharmacia, Uppsala,Sweden). Eluted MAb is preferably concentrated and dialyzed againstsaline. The concentration of the antibody may be determined usingabsorbance of light at 280 nm. Monoclonal antibodies can be isotypedusing the Mouse MonAB ID KIT (HRP) (Zymed, San Francisco, Calif.).Biotinylated MAB (b-mAb) were generated for each clone as described by(Harriman, G. G., In: Current Protocols in Immunology, vol. 1., Coligan,J. E. et al., eds., Greene Publishing Associates and Wiley-Interscience,New York, N.Y., p. 6.5.1 (1991)) for use in ELISA.

III. The Molecules of the Present Invention

The present invention concerns recombinantly produced human IL-5, aswell as mutants and mimetics of IL-5. The invention also concernsantibodies, and binding molecules that are capable of specific bindingto IL-5. The characteristics of these molecules are discussed below.

A. Antibodies and Binding Molecules Capable of Specifically Binding withhIL-5

The present invention concerns the production and use of molecules thatare capable of "specific binding" to one another. As used herein, amolecule is said to be capable of "specific binding" to anothermolecule, if such binding is dependent upon the respective structures ofthe molecules. The known capacity of an antibody to bind to an immunogenis an example of "specific binding." Such interactions are in contrastto non-specific binding that involve classes of compound, irrespectiveof their chemical structure (such as the binding of proteins tonitrocelulose, etc.) Most preferably, the antibody and other bindingmolecules of the present invention will exhibit "highly specificbinding," such that they will be incapable of substantially incapable ofbinding to closely related heterologous molecules. Indeed, the preferredmonoclonal antibodies of the present invention exhibit the capacity tobind to human IL-5, but are substantially incapable of binding murineIL-5; such antibodies are capable of highly specific binding to humanIL-5, as those terms are used herein.

The above-described divalent antibody molecules (i.e. possessing twoIL-5 binding domains) comprise one class of the immunoglobulin reagentsof the present invention, however, the invention also includesderivatives and modified immunoglobulins that have the capacity tospecifically bind to human IL-5.

Thus, in one embodiment, such molecules will comprise fragments (such as(F(ab'), F(ab')₂) that are produced, for example, by the proteolyticcleavage of the mAbs, or single-chain immunoglobulins producible, forexample, via recombinant means. Such antibody derivatives aremonovalent. In one embodiment, such fragments can be combined with oneanother, or with other antibody fragments or receptor ligands to form"chimeric" binding molecules. Significantly, such chimeric molecules maycontain substituents capable of binding to different epitopes of thesame molecules (such as two different IL-5 epitopes), or they may becapable of binding to an IL-5 epitope and a "non-IL-5" epitope. In oneembodiment, such "non-IL-5" epitopes are selected such that the chimericmolecule can bind to cellular receptors, such as hormone receptors,immune response receptors, etc. Because such a molecule can bind toIL-5, it may be used to "ferry" IL-5 to any cell that arrays suchcellular receptors, regardless of whether the cell expresses the IL-5receptor.

In one embodiment, any of the above-described molecules can be labelled,either detectably, as with a radioisotope, a paramagnetic atom, afluorescent moiety, an enzyme, etc. in order to facilitate the itsdetection in, for example, in situ or in vivo assays. In an alternativeembodiment, the molecules can be labelled with a toxin molecule in orderto provide a cell receptor specific targeting of the toxin. Themolecules may be labelled with reagents such as biotin, in order to, forexample, facilitate their recovery, and/or detection.

The monoclonal antibodies of the present invention have been found tocomprise two antigenic groups, and to thereby establish that human IL-5contains multiple epitopes that can be recognized by murine splenocytes.The specificity of these antibodies to hIL-5 was assessed by directELISA. All mAb bound only rhIL-5. No binding was detected withbaculovirus-expressed murine IL-5 indicating that the observedanti-hIL-5 mAb binding was not specific to Sf9 cell glycosylation of therhIL-5.

By competitive ELISA, all mAb could be divided between two bindinggroups, A and B. Group A consisted of eleven mAb and group B consistedof four mAb which competed for TRFK-5 binding to hIL-5. No furtherdistinction could be made within a given group, as competition could bea result of epitope identify or merely steric interference between agiven pair of mAb within each group. However since TRFK-5 is capable ofbinding mIL-5, its epitope cannot be the same as any recognized by anygroup B mAb. Thus competition for binding between TRFK-5 and group B mAbmust be a result of steric hinderance. Therefore, at least threeepitopes have been demonstrated for hIL-5, all of which areneutralizing. Through ELISA and Western analysis these mAb have beenfurther characterized and subdivided, and found to define two epitopesof human IL-5. The isolation of the antibodies permits the design offragments and derivatives.

Where the antibodies or their fragments are intended for therapeuticpurposes, it may desirable to "humanize" them in order to attenuate anyimmune reaction. Humanized antibodies may be produced, for example byreplacing an immunogenic portion of an antibody with a corresponding,but non-immunogenic portion (i.e. chimeric antibodies) (Robinson, R. R.et al., International Patent Publication PCT/US86/02269; Akira, K. etal., European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison, S. L. et al., European Patent Application173,494; Neuberger, M. S. et al., PCT Application WO 86/01533; Cabilly,S. et al., European Patent Application 125,023; Better, M. et al.,Science 240:1041-1043 (1988); Liu, A. Y. et al., Proc. Natl. Acad. Sci.USA 84:3439-3443 (1987); Liu, A. Y. et al., J. Immunol. 139:3521-3526(1987); Sun, L. K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987);Nishimura, Y. et al., Canc. Res. 47:999-1005 (1987); Wood, C. R. et al.,Nature 314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst.80:1553-1559 (1988); all of which references are incorporated herein byreference). General reviews of "humanized" chimeric antibodies areprovided by Morrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi,V. T. et al., Bio Techniques 4:214 (1986); which references areincorporated herein by reference).

Suitable "humanized" antibodies can alternatively be produced by CDR orCEA substitution (Jones, P. T. et al., Nature 321:552-525 (1986);Verhoeyan et al., Science 239:1534 (1988); Beidler, C. B. et al., J.Immunol. 141:4053-4060 (1988); all of which references are incorporatedherein by reference).

B. hIL-5 Mutants and Mimetic Agents

The identification of the monoclonal antibodies of the present inventionpermits the design and development of hIL-5 mutants and mimetic agents.Such agents can be used to treat inflammatory diseases such asthma;adult respiratory distress syndrome (ARDS) or multiple organ injurysyndromes secondary to septicemia or trauma; reperfusion injury ofmyocardial or other tissues; acute glomerulonephritis; reactivearthritis; dermatoses with acute inflammatory components; eosinophilia;acute purulent meningitis or other central nervous system inflammatorydisorders; thermal injury; hemodialysis; leukapheresis; ulcerativecolitis; Crohn's disease; necrotizing enterocolitis; granulocytetransfusion associated syndromes; cytokine-induced toxicity, as well asaberrant responses of the specific defense system include the responseto antigens such as rubella virus, autoimmune diseases, delayed typehypersensitivity response mediated by T-cells.

In particular, the epitopes of hIL-5 recognized by the antibodies of thepresent invention define sites of probable importance to the structureor functions of IL-5. Since the cDNA sequence that encodes hIL-5 isknown, it is possible to mutate or alter that sequence via classical orrecombinant means. Such mutated gene sequences can be expressed andevaluated for their capacity to bind the antibodies of the presentinvention. In this manner, mutant IL-5 species can be isolated andevaluated for their capacity to inhibit IL-5-mediated inflammation, orother IL-5 functions. Similarly, such species can be evaluated for theircapacity to induce a hyper inflammatory response, such as would bedesirable in immune compromised or immune suppressed individuals (forexamples, AIDS or ARC patients).

In one embodiment, such molecules are derived via mutagenesis of IL-5,or from polypeptide fragments of the IL-5 molecule. Such fragments canbe prepared via chemical or recombinant means (such as by randomlycleaving IL-5-encoding DNA and then incorporating the cleavage fragmentsinto translatable expression vectors). In this manner, a library ofdifferent peptide fragments can be obtained and evaluated. Thebiological activity of the polypeptides can be assessed by theimmunoassays described herein, or by the capacity of the molecules tobind to anti-IL-5 antibody columns.

IL-5 mutant analogs can be identified either rationally, as describedbelow, or via established methods of mutagenesis (see, for example,Watson, J. D. et al., Molecular Biology of the Gene, Fourth Edition,Benjamin/Cummings, Menlo Park, Calif. (1987). Significantly, a randommutagenesis approach requires no a priori information about the genesequence that is to be mutated. This approach has the advantage that itassesses the desirability of a particular mutant on the basis of itsfunction, and thus does not require an understanding of how or why theresultant mutant protein has adopted a particular conformation. Indeed,the random mutation of target gene sequences has been one approach usedto obtain mutant proteins having desired characteristics (Leatherbarrow,R. J. Prot. Eng. 1:7-16 (1986); Knowles, J. R., Science 236:1252-1258(1987); Shaw, W. V., Biochem. J. 246:1-17 (1987); Gerit, J. A. Chem.Rev. 87:1079-1105 (1987)). Alternatively, where a particular sequencealteration is desired, methods of site-directed mutagenesis can beemployed. Thus, such methods may be used to selectively alter only thoseamino acids of the protein that are believed to be important (Craik, C.S., Science 228:291-297 (1985); Cronin, C. S. et al., Biochem.27:4572-4579 (1988); Wilks, H. M. et al., Science 242:1541-1544 (1988)).The analysis of such mutants can also be facilitated through the use ofa phage display protein ligand screening system (Lowman, H. B. et al.,Biochem. 30:10832-10838 (1991); Markland, W. et al., Gene 109:13-19(1991); Roberts, B. L. et al., Proc. Natl. Acad. Sci. (U.S.A.)89:2429-2433 (1992); Smith, G. P., Science 228:1315-1317 (1985); Smith,R. P. et al., Science 248:1126-1128 (1990), all herein incorporated byreference)). In general, this method involves expressing a fusionprotein in which the desired protein ligand is fused to the C-terminusof a viral coat protein (such as the M13 Gene III coat protein, or alambda coat protein).

The antibodies of the present invention can alternatively be used toscreen molecules other than IL-5 (or IL-5 fragments) for their capacityto mimic one or more functions of IL-5. Such functions include not onlythe biological activities of the molecule, but also the capacity of IL-5to bind to its normal cellular receptor molecule. Indeed, mimetic agentsthat are capable of binding to the IL-5 receptor, but are substantiallyincapable of mediating other IL-5 functions are highly desired. Thepresent invention provides a means for obtaining such molecules.

Thus, the present invention concerns mimetic analogs of IL-5. As usedherein, a "mimetic analog" of hIL-5 is a molecule that retains abiological activity of the molecule, but will typically be unrelatedchemically. An organic molecule whose structure mimics the active site,or a binding site of hIL-5 would comprise a "mimetic analog" of thatprotein. The hIL-5 mimetic agents of the present invention may be anoligonucleotides, a proteinaceous compound (including both glycosylatedand non-glycosylated proteins), or a non-proteinaceous compound (such asa steroid, a glycolipid, etc.) provided that the agent mimics a functionof either an entire hIL-5 nucleic acid molecule, or a fragment thereof.Preferred classical analogs include polypeptides (including circular aswell as linear peptides) whose sequences comprise the active catalyticor binding sites of an hIL-5 protein, or oligonucleotide fragments ofnucleic acid hIL-5 molecules that are capable of either repressing orinducing hIL-5 activity. Preferred mimetic analogs include polypeptidesthat are not fragments of an hIL-5 protein, or mutants thereof, butnevertheless exhibit a capacity to induce senescence in an hIL-5-likemanner, or to induce cellular proliferation in the manner of an hIL-5antagonist.

Mimetic analogs of naturally occurring hIL-5 molecules may be obtainedusing the principles of conventional or of rational drug design(Andrews, P. R. et al., In: Proceedings of the Alfred Benzon Symposium,volume 28, pp. 145-165, Munksgaard, Copenhagen (1990); McPherson, A.Eur. J. Biochem. 189:1-24 (1990); Hol, W. G. J. et al., In: MolecularRecognition: Chemical and Biochemical Problems, Roberts, S. M. (ed.);Royal Society of Chemistry; pp. 84-93 (1989); Hol, W. G. J.,Arzneim-Forsch. 39:1016-1018 (1989); Hol, W. G. J., Agnew, Chem. Int.Ed. Engl. 25:767-778 (1986) all herein incorporated by reference).

In accordance with the methods of conventional drug design, the desiredmimetic molecules are obtained by randomly testing molecules whosestructures have an attribute in common with the structure of a "native"hIL-5 molecule, or a molecule that interacts with an hIL-5 molecule. Thequantitative contribution that results from a change in a particulargroup of a binding molecule can be determined by measuring the capacityof competition or cooperatively between the native hIL-5 molecule andthe putative mimetic.

In one embodiment of rational drug design, the mimetic is designed toshare an attribute of the most stable three-dimensional conformation ofan hIL-5 molecule. Thus, the mimetic analog of a hIL-5 molecule may bedesigned to possess chemical groups that are oriented in a waysufficient to cause ionic, hydrophobic, or van der Waals interactionsthat are similar to those exhibited by the hIL-5 molecule. In a secondmethod of rational design, the capacity of a particular hIL-5 moleculeto undergo conformational "breathing" is exploited. Such"breathing"--the transient and reversible assumption of a differentmolecular conformation--is a well appreciated phenomenon, and resultsfrom temperature, thermodynamic factors, and from the catalytic activityof the molecule. Knowledge of the 3-dimensional structure of the hIL-5molecule (Milburn, M. W. et al., Nature 363:172-176 (1993); hereinincorporated by reference) facilitates such an evaluation. An evaluationof the natural conformational changes of an hIL-5 molecule facilitatesthe recognition of potential hinge sites, potential sites at whichhydrogen bonding, ionic bonds or van der Waals bonds might form or mightbe eliminated due to the breathing of the molecule, etc. Suchrecognition permits the identification of the additional conformationsthat the hIL-5 molecule could assume, and enables the rational designand production of mimetic analogs that share such conformations.

The preferred method for performing rational mimetic design employs acomputer system capable of forming a representation of thethree-dimensional structure of the hIL-5 molecule (such as thoseobtained using RIBBON (Priestle, J., J. Mol. Graphics 21:572 (1988)),QUANTA (Polygen), InSite (Biosyn), or Nanovision (American ChemicalSociety). Such analyses are exemplified by Hol, W. G. J. et al. (In:Molecular Recognition: Chemical and Biochemical Problems, Roberts S. M.(ed.); Royal Society of Chemistry; pp. 84-93 (1989), Hol, W. G. J.(Arzneim-Forsch. 39:1016-1018 (1989)), and Hol, W. G. J., Agnew. Chem.Int. Ed. Engl. 25:767-778 (1986)).

IV. The Uses of the Molecules of the Present Invention

The anti-hIL-5 antibodies of the present invention can be used toisolate, characterize, and quantitate hIL-5. They may also be used toeffect the precise mapping of those hIL-5 epitopes critical for bindingto the IL-5 receptor (Limaye, A. P., et al., J. Clin. INvest. 88:1418(1991)). Moreover, since anti-mIL-5 monoclonal antibodies can blockmIL-5 mediated eosinophilia in both mice and guinea pigs (Bloom, J. W.,et al., J. Immunol. 151:2707 (1993); Coffman, R. L., et al., Science245:308 (1989); Rennick, D. M., et al., Blood 76:312 (1990); Owen, W.F., et al., J. Exp. Med. 170:343 (1989); Chand, N., et al., Eur. J.Pharm 211:121 (1991)), the anti hIL-5 mAb of the present invention hasutility in blocking IL-5 mediated inflammation in humans.

A. Immunoassay Analysis of hIL-5 Expression

The present invention provides a highly sensitive immunoassay suitablefor detecting and/or quantitating the presence of human interleukin-5 ina sample. As used herein, the term "sample" is intended to encompassbiological specimens derived from a human or other animal source (suchas, for example, blood, stool, sputum, mucus, serum, urine, saliva,teardrop, a biopsy sample, an histology tissue sample, a PAP smear,etc.) including samples derivated a cellular preparation (such as acellular extract, lysate, cytosol, etc.). As will be understood, thesample may need to be diluted with buffer, or concentrated (as with anevaporator or lyophilizer) in order to ensure that the amount of IL-5contained in the sample is within the detection limits of the assay.

As will be understood from the well-known principles of immunoassays,alternative formats, such as immunometric assays (also known as a"two-site" or "sandwich" assays), including both "forward,""simultaneous" and "reverse" assays. For example, in a "forward" assays,antibody would be bound to a solid support (such as a microtiter plate,test tube, dipstick, etc.), and then first contacted with the samplebeing tested to extract the hIL-5 from the sample by formation of abinary solid phase antibody-hIL-5 complex. After incubation and washing,the support would be placed in contact with a quantity of labeledantibody specific for hIL-5 (which functions as a "reporter"). After asecond incubation period to permit the labeled antibody to complex withthe hIL-5 bound to the solid support through the unlabeled antibody, thesolid support would be washed a second time to remove the unreactedlabeled antibody. This type of forward sandwich assay may be a simple"yes/no" assay to determine whether hIL-5 is present or may be madequantitative by comparing the amount of retained labeled antibody withthat obtained for a standard sample containing known quantities ofhIL-5. Such "two-site" or "sandwich" assays are described by Wide, In:Radioimmune Assay Method, (Kirkham et al., Ed.), E. & S. Livingstone,Edinburgh, pp 199-206 (1970), herein incorporated by reference).

In a "simultaneous" assay, a single incubation step is employed in whichthe bound antibody and the labeled antibody are both added to the samplebeing tested at the same time. After the incubation is completed, thesolid support is washed to remove the residue of fluid sample anduncomplexed labeled antibody. The presence of labeled antibodyassociated with the solid support is then determined as it would be in aconventional "forward" sandwich assay.

In a "reverse" assay, a solution of labeled antibody is incubated withthe fluid sample followed. After such incubation, the mixture is placedin contact with a solid support to which unlabeled antigen has beenpreviously bound. After a second incubation, the solid phase is washedin a conventional fashion to free it from the residue of the samplebeing tested and the solution of unreacted labeled antibody. Thedetermination of labeled antibody associated with a solid support isthen determined as in the "simultaneous" and "forward" assays.

In its most preferred embodiment, the ELISA of the present inventionemploys an anti-hIL-5 monoclonal antibody. Most preferably, suchantibodies are generated by immunizing a heterologous mammal (such as amouse, rat, rabbit, etc.) with hIL-5, and then harvesting the splenicleukocytes of the animal, and fusing them with a suitable myeloma cell,in the manner described above. In one embodiment, such monoclonalantibodies can be directly employed in an immuoassay format.Alternatively, such antibodies may be cleaved or processed to formfragments that retain the capacity to bind hIL-5. Examples of suchfragments include (F(ab'), F(ab')₂ fragments. The fragments can bedirectly used to assay IL-5 expression, or they can be combined withfragments of other antibodies in order to form non-naturally occurring"divalent" or "multivalent" antibodies. Such antibodies could, forexample, therefore possess the capacity to bind to multiple epitopes ofIL-5, or to an epitope of IL-5 and an epitope of another molecule.

Suitable solid supports may be composed, for example, of materials suchas glass, paper, polystyrene, polypropylene, polyethylene, dextran,nylon, amylases, natural and modified celluloses, polyacrylamides,agaroses, or magnetite. The nature of the support can be either solubleto some extent or insoluble for the purposes of the present invention.The support material may have virtually any possible structuralconfiguration so long as the bound IL-5 is capable of binding to ananti-IL-5 antibody. Thus, the support configuration may be spherical, asin a bead, or cylindrical, as in the inside surface of a test tube, orthe external surface of a rod. Alternatively, the surface may be flatsuch as a sheet, test strip, etc. Those skilled in the art will notemany other suitable carriers for binding monoclonal antibody, or will beable to ascertain the same by use of routine experimentation. Mostpreferably, the support will be a polystyrene microtiter plate.

B. Antibody-Enhanced Purification or Recovery of IL-5

The antibodies of the present invention can be employed to enable arapid recovery of substantially pure IL-5 from either recombinant ornatural sources (e.g. serum). Thus, such molecules can be used in largescale affinity chromatography to effect the removal of IL-5 from asupernatant. Significantly, since the antibodies of the presentinvention do not recognize murine IL-5, they are particularly suitablefor use in purifying human IL-5 from sources that may be contaminatedwith the murine IL-5 homolog.

Thus, the antibodies of the present invention can be bound to a resin,such as sepharose. The treated resin can be incubated, either in batch,as a slurry, or, more preferably, as a continuous throughput column inorder to effect such recovery.

C. IL-5 Mimetic Therapy

As indicated, one aspect of the present invention concerns therapeuticmolecules that are capable of modulating the eosinophilic responseinduced by IL-5, and thus of attenuating an undesired inflammatoryresponse. Conversely, The antibodies of the present invention can beused to obtain hyperinflammatory analogs of IL-5 that can be used inimmune suppressed individuals.

Such therapeutic molecules of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby these materials, or their functional derivatives,are combined in admixture with a pharmaceutically acceptable carriervehicle. Suitable vehicles and their formulation, inclusive of otherhuman proteins, e.g., human serum albumin, are described, for example,in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack.Easton, Pa. (1980)). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of an antisense oligonucleotide, or itsequivalent, or their functional derivatives, together with a suitableamount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb an antisense oligonucleotide,or its equivalent, or their functional derivatives. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine, sulfate) and the concentration of macromolecules as well asthe methods of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate an antisense oligonucleotide, or itsequivalent, or their functional derivatives, into particles of apolymeric material such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively,instead of incorporating these agents into polymeric particles, it ispossible to entrap these materials in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcelluose or gelatine-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

The therapeutic compositions of the present invention can also beformulated for administration parenterally by injection, rapid infusion,nasopharyngeal absorption (intranasopharangeally), dermabsorption, ororally. The compositions may alternatively be administeredintramuscularly, or intravenously. Compositions for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Carriers, adjucts orocclusive dressings can be used to increase tissue permeability andenhance antigen absorption. Liquid dosage forms for oral administrationmay generally comprise a liposome solution containing the liquid dosageform. Suitable forms for suspending liposomes include emulsions,suspensions, solutions, syrups, and elixirs containing inert diluentscommonly used in the art, such as purified water. Besides the inertdiluents, such compositions can also include wetting agents, emulsifyingand suspending agents, or sweetening, flavoring, coloring or perfumingagents.

A therapeutic composition is said to be "pharmacologically acceptable"if its administration can be tolerated by a recipient patient. Such anagent is said to be administered in a "therapeutically effective amount"if the amount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

Generally, the dosage needed to provide an effective amount of thecomposition will vary depending upon such factors as the recipient'sage, condition, sex, and extent of disease, if any, and other variableswhich can be adjusted by one of ordinary skill in the art.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified. In the Examples that follow,unless otherwise stated, all values represent mean of triplicate samples±standard error of the mean. The Tukey test was used to compare groups.Significance was assigned by a P value of less than 0.05.

EXAMPLE 1 HIL-5 CLONING AND EXPRESSION

A hIL-5 cDNA containing plasmid [ph IL5 115.1] was obtained from theATCC [#59394]. The original cDNA was isolated and cloned into pcDVIvector by Yokata, T., et al. (Proc. Natl. Acad. Sci. U.S.A., 84:7388(1987)) using a homopolymeric tailing method. This insert including G-Ctails was excised from its original vector and inserted in to the BamH1site of PBR 322 by the ATCC. To maximize efficiency of expression, weused PCR to insert a new BamHI site 30 bases upstream from the IL-5start codon and recloned the insert into the BamHI site of baculovirustransfer vector, PVL 941. The PCR priming sequence [SEQ ID NO: 1GAGGATCCAAAGGCAAACGCAGA] was chosen to place the hIL-5 start codon outof frame and 70 bases downstream of the mutated polyhedron start codon,ATT. The insert was sequenced in PVL 941 to insure sequence fidelity.The recombinant transfer vector, PVL-hIL-5, was cotransfected with wildtype AcMNPV into the Sf9 cell line and recombinant viruses picked andsubcloned by plaque assay and hybridization using standard methodology(Summers, M. D., et al., A manual of methods for baculovirus vector andinsect cell culture procedures. The Texas Agricultural ExperimentStation, College Station, Tex. (1987)). Recombinant hIL-5 (rhIL-5) wasproduced in 400 ml spinner flasks of Sf9 cells using an MOI of ten.After five days of infection, the cell culture supernate was filteredand made to 0.5% NP-40 and 0.05% tween 20 and run over a CnBr-Sepharosecolumn derivatized with the anti-mIL-5 mAB, TRFK-5 (Mita, S., et al., J.of Immunol. Meth. 125:233 (1987)). The column was run essentiallyaccording to the method of Schumacher, J. H., et al., (J. Immuno.141-1576 (1988)). Purified rhIL-5 was tested for purity by silverstaining following SDS-PAGE and was quantiated using the microliter BCAprotein assay (Pierce, Rockford, Ill.) and by ELISA using TRFK-5 in adouble antibody sandwich as described (Mita, S., et al., J. of Immunol.Meth. 125:233 (1987))). Functional quantitation was done using themurine BCL, proliferation assay (Mita, S., et al., J. Immunol. Meth.125:233 (1987)).

EXAMPLE 2 MONOCLONAL ANTIBODY GENERATION

BALB/c mice were immunized with 25 μg of affinity purified baculovirusderived rhIL-5 emulsified 1:1 in TiterMax adjuvant (Vaxcel, Norcross,Ga.) at two intramuscular sites, one intraperitoneal site, and onesubcutaneous site at the base of the tail. An additional i.v. injectionof 25 μg rhIL-5 was given in normal saline three weeks later. Mice werebled 11 days following the second injection and screened from anti-hIL-5antibodies in a direct binding ELISA. The mouse with the highestanti-hIL-5 titer was given a third i.v. injection of 25 μg rhIL-5. Threedays later splenic leukocytes were harvested and fused with P3X63Ag8.653myeloma cells, using polyethylene glycol. After fusion, cells wereplated on five 96 wells plates in 100 μ1/well HAT selection media andincubated at 37° C. in 5% CO₂ air. After one week, all wells were screenfor anti-hIL-5 activity again by direct binding ELISA and a selectnumber of positive wells subcloned by three rounds of limitingdilutions. Fifteen clones were expanded stepwise to a T75 flask(Corning, Corning, N.Y.). Nude mice were primed with 0.5 ml of2,6,10,14-tetramethypentadecane (Aldrich, Milwaukee, Wis.). After 5days, each clone was harvested, pelleted, and resuspended in sterile PBSto a final density of 2.5×10⁶ cell/ml. For all fifteen clones a pair ofnude mice were injected i.p. using a 22-G needle with 5.0×10⁶ cell.Ascites was collected daily by drainage using a 18-G needle and pooledfor batch processing. Ascites was spun at 1000×g and the supernatantcollected and lipocleaned with Seroclear (Calbiochem, San Diego, Calif.)following vendor specifications. Murine antibody was purified using aGammaBind Plus Sepharose column (Pharmacia, Uppsala, Sweden). Eluted MAbwas concentrated and dialyzed against 0.15M NaCl. Concentration wasdetermine using absorbance of light at 280 nm. Monoclonal antibodieswere isotyped using the Mouse MonoAB ID Kit (HRP) (Zymed, San Francisco,Calif.). Biotinylated MAB (b-mAb) were generated for each clone aspreviously described (Harriman, G. R., In: Current Protocols inImmunology, vol. 1., Coligan, J. E. et al., eds., Greene PublishingAssociates and Wiley-Interscience, New York, N.Y., p. 6.5.1 (1991)) foruse in ELISA.

To assay for the production of anti-hIL-5 antibodies, polystyrene ELISAplates (Corning, Corning, N.Y.) were coated with either 50 μl of rhIL-5or rmIL-5 (Harriman, G. R., In: Current Protocols in Immunology, vol.1., Coligan, J. E. et al., eds., Greene Publishing Associates andWiley-Interscience, New York, N.Y., p. 6.5.1 (1991)) at a concentrationof 2 μg/ml in PBS and incubated overnight at 4° C. Plates were blockedwith PBS 0.5% tween 10% calf serum. Fifty microliters of eachbiotinylated-mAb (b-mAb) at a 1/250 dilution in blocker plus a controlwithout mAb were incubated. Fifty microliters of strepavidin-alkalinephosphatase (Molecular Probes, Inc., Eugene, Oreg.) at 1/500 dilution inblocker was incubated. Finally 100 μl of a 4-methylumbelliferylphosphate(MUP) (Sigma, St. Louis, Mo.) substrate solution (0.05 mg MUP/ml in 1 Mdiethanolamine 1 mM MgCl₂ pH 9.8) was added and placed on a shaker for30 minutes at room temperature. Fluorescence was read on a TitertekFluoroskan II fluorometer (Flow Laboratories, McLean, Va.) at 30 and 45minutes). Fluorometric values were recorded using Titersoft II E.I.A.Software (Flow Laboratories, McLean Va.). Unless otherwise stated allincubations were done on a shaker at room temperature for one hour.Between each incubation step plates were washed five times with PBS 0.5%tween.

Following three rounds of limiting-dilution-cloning, fifteen hybridomasproducing anti-rhIL-5 mAb were selected and given the designation IL5.1through IL5.15. The hybridomas designated as IL5.1 through IL5.15 weredeposited with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, on Jan. 13, 1999. IL5.1was assigned the ATCC designation number HB-12637, and is referenced bythe depositor as Murine hybridoma 5.1.1; IL5.2 was assigned ATCC numberHB-12627, and is referenced as Murine hybridoma h5.1.2; IL5.3 wasassigned ATCC number HB-12640, and is referenced as Murine hybridomah5.1.3; IL5.4 was assigned ATCC number HB-12626, and is referenced asMurine hybridoma h5.2.1; IL5.5 was assigned ATCC number HB-12634, and isreferenced as Murine hybridoma h5.2.2; IL5.6 was assigned ATCC numberHB-12636, and is referenced as Murine hybridoma h5.2.3; IL5.7 wasassigned ATCC number HB-12638, and is referenced as Murine hybridoma5.2.4; IL5.8 was assigned ATCC number HB-12633, and is referenced asMurine hybridoma h5.3.1; IL5.9 was assigned ATCC number HB-12628, and isreferenced as Murine hybridoma h5.3.2; IL5.10 was assigned ATCC numberHB-12630, and is referenced as Murine hybridoma h5.4; IL5.11 wasassigned ATCC number HB-12635, and is referenced as Murine hybridomah5.5; IL5.12 was assigned ATCC number HB-12639, and is referenced asMurine hybridoma h5.6; IL5.13 was assigned ATCC number HB-12631, and isreferenced as Murine hybridoma h5.7.1; IL5.14 was assigned ATCC numberHB-12629, and is referenced as Murine hybridoma h5.7.2; and, IL5.15 wasassigned ATCC number HB-12632, and is referenced as Murine hybridomah5.7.3. All were of the isotype IgG₁, kappa. Each mAb was isolated fromascites fluid over protein G columns for determination of bindingcharacteristics.

Using the previously characterized rat mAb, TRFK-5 (43), as a positivecontrol, each murine mAb was screened by direct ELISA for binding torhIL-5 and rmIL-5 (Table I). All anti-hIL-5 mAb bound selectively torhIL-5 and lacked any binding to rmIL-5. In Table I, values are meanfluorescence units ±1; TRFK-5 is a positive control mAb for binding toboth hIL-5 and mIL-5.

                  TABLE I                                                         ______________________________________                                        Binding of mAb to IL-5 By Direct ELISA                                          mAb             rhIL-5    rmIL-5                                            ______________________________________                                        IL5.1         6292 ± 110                                                                           0 ± 5                                                IL5.2  6012 ± 58  0 ± 5                                                 IL5.3  6028 ± 115  0 ± 14                                               IL5.4  6020 ± 119 0 ± 6                                                 IL5.5  6096 ± 113 0 ± 6                                                 IL5.6  6106 ± 137 0 ± 4                                                 IL5.7  6138 ± 110 0 ± 5                                                 IL5.8  6374 ± 85  0 ± 5                                                 IL5.9  6217 ± 62  0 ± 3                                                 IL5.10 5879 ± 35  0 ± 5                                                 IL5.11 5895 ± 20  0 ± 4                                                 IL5.12 6015 ± 14   0 ± 31                                               IL5.13 5955 ± 8  0 ± 5                                                  IL5.14 5883 ± 36  0 ± 6                                                 IL5.15 5812 ± 2  0 ± 3                                                  TRFK-5 6392 ± 122 5916 ± 1                                              no MAb  0 ± 1 0 ± 1                                                   ______________________________________                                    

Thus, a panel of fifteen mAb were generated against baculovirusexpressed rhIL-5. All mAb were of the isotype IgG¹, Kappa. All werespecific for hIL-5 and capable of neutralizing hIL-5 biologicalactivity. Through ELISA and Western analysis these mAb have been furthercharacterized and subdivided. Although they have already been provenuseful for isolating, characterizing, and quantitating hIL-5, theyshould facilitate precise mapping of hIL-5 epitopes critical for bindingto the IL-5 receptor (Limaye, A. P., et al., J. Clin. INvest. 88:1418(1991)). The ability of mAb to block IL-5 mediated eosinophila in bothmice and guinea pigs (Bloom, J. W., et al., J. Immunol. 151:2707 (1993);Coffman, R. L., et al., Science 245:308 (1989); Rennick, D. M., et al.,Blood 76:312 (1990); Owen, W. F., et al., J. Exp. Med. 170:343 (1989);Chand, N., et al., Eur. J. Pharm. 211:121 (1991)), suggests thatanalogous therapy with anti-hIL-5 mAb might be feasible in blocking IL-5mediated inflammation in humans.

EXAMPLE 3 COMPETITIVE BINDING ELISA

Differences in rhIL-5 binding among the mAb were assessed by competitiveELISA. Binding of each b-mAb was completed with a 100-fold excess ofeach unlabeled mAb.

Polystyrene ELISA plates (Corning, Corning, N.Y.) were coated with 50 μlof rhIL-5 (2 μg/ml in PBS) and incubated overnight at 4° C. Plates wereblocked with PBS 0.5% tween 10% calf serum. To assess competitivebinding, 50 μl in blocker) of each unlabeled mAb was preincubated in ablock of wells. Buffer without mAb was added to a block of controlwells. Without washing, fifty microliters of each b-mAb (0.25 μg/ml inblocker) were then added to a pair of wells in each block and incubatedfor another hour at room temperature. The same strepavidin-alkalinephosphatase/MUP detection system described above was used to determinethe degree of b-mAb binding.

As shown in Table II, all mAb could be divided into one of two ELISAbinding groups. Group A consisted of mAb IL5.1 through IL5.11. Group Bconsisted of IL5.12 through IL5.15. TRFK-5 was also found to compete forbinding with all group B anti-hIL-5 mAb and was thus assigned to thatbinding group. Results were represented as percent inhibition of maximumbinding, defined as the signal generated by each b-mAb in the absence ofany competing mAb. A given b-mAb competing with itself represented themaximum inhibition achievable.

                                      TABLE II                                    __________________________________________________________________________    Analysis of anti-hIL-5 mAb binding by competitive ELISA.sup.a                       Competing mAb                                                           Biotinylated                                                                        Group A                              Group B                            mAb   IL5.1                                                                            IL5.2                                                                            IL5.3                                                                            IL5.4                                                                            IL5.5                                                                            IL5.6                                                                            IL5.7                                                                            IL5.8                                                                             IL5.9                                                                             IL5.10                                                                            IL5.11                                                                            IL5.12                                                                            IL5.13                                                                            IL5.14                                                                            IL5.15                                                                            TRFK-5             __________________________________________________________________________    b-IL5.1                                                                             94 94 93 96 97 96 97 95  94  94  97  0   0   0   0   0                    b-IL5.2 95 96 95 97 98 98 98 96 94 96 97 0 0 0 0 0                            b-IL5.3 95 95 94 95 98 98 98 95 93 95 96 0 0 0 0 0                            b-IL5.4 80 80 79 88 90 90 91 83 76 83 88 0 0 0 0 0                            b-IL5.5 81 84 83 90 92 93 93 87 79 86 90 0 0 0 0 0                            b-IL5.6 77 80 79 87 90 90 90 81 75 81 84 0 0 0 0 0                            b-IL5.7 86 88 86 93 94 94 94 88 85 90 94 0 0 0 0 0                            b-IL5.8 94 95 93 98 94 97 98 95 93 95 97 0 0 0 0 0                            b-IL5.9 94 95 94 98 98 98 98 95 95 96 98 0 0 0 0 0                            b-IL5.10 72 74 74 75 74 74 75 76 70 85 79 0 0 0 0 0                           b-IL5.11 73 75 72 86 87 88 87 78 68 79 84 0 0 0 0 0                           b-IL5.12  0  0  0  0  0  0  0  0  0  0  0 97  88  85  91  78                  b-IL5.13  0  0  0  0  0  0  0  0  0  0  0 96  96  96  96  83                  b-IL5.14  0  0  0  0  0  0  0  0  0  0  0 96  95  96  96  82                  b-IL5.15  0  0  0  0  0  0  0  0  0  0  0 96  94  96  96  81                  b-TRFK-5  0  0  0  0  0  0  0  0  0  0  0 88  85  86  87  94                __________________________________________________________________________     Values represent percent inhibition of maximum bmAb signal by a hundred       fold excess of competing mAb.                                                 Maximum signal is defined as that generated by a given bmAb in the absenc     of competing mAb.                                                             Values in bold indicate inhibition of each bmAb by unlabeled self.       

In sum, specificity to hIL-5 was assessed by direct ELISA. All mAb boundonly rhIL-5. No binding was detected with baculovirus expressed mIL-5indicating that anti-hIL-5 mAb binding was not specific to Sf9 cellglycosylation of the rhIL-5. By competitive ELISA, all mAb could bedivided between two binding groups, A and B (Table II). Group Aconsisted of eleven mAb and group B consisted of four mAb which competedfor TRFK-5 binding to hIL-5. No further distinction could be made withina given group, as competition could be a result of epitope identify ormerely steric interference between a given pair of mAb within eachgroup. However since TRFK-5 is capable of binding mIL-5 (Table I), itsepitope cannot be the same as any recognized by any group B mAb. Thuscompetition for binding between TRFK-5 and group B mAb must be a resultof steric hinderance. Therefore, at least three epitopes have beendemonstrated for hIL-5, all of which are neutralizing.

EXAMPLE 5 BCL₁ PROLIFERATION ASSAY

All mAb were screened for their ability to neutralize rhIL-5 (1 μg/ml)and rmIL-5 (1 ng/ml) biological activity in a BCL₁ proliferation assaywith a constant concentration of IL-5 (FIG. 1).

For this purpose, 25 μl of rhIL-5 (1 μg/ml final concentration) orrmIL-5 (1) ng/ml final concentration) were preincubated with 25 μl ofeach mAb, a positive control mAb, TRFK-5, or an irrelevant isotypematched mAb (OKT11), all at a final concentration of 20 μg/ml, or withmedia only for one hour at 37° C. Fifty microliters of BCL₁ cells (4×10⁵cells/ml) in RPMI media, 10% FCS, 2 mM L-glutamine, 50 μg/ml 2-ME, 15 mMHEPES, and 50 μg/ml gentamicin were added and incubate for 16 hours at37° C. in 5% CO₂ humidified air. After 16 hours, 1 μCi of [³ H]thymidine(Amersham Buckinghamshire, England) was added per well and incubated foranother 8 hours. At 24 hours the cells were harvested and [³ H]thymidineincorporation measured by a liquid scintillation counter (Beckman, PalaAlto, Calif.).

To assess the dose dependency of anti-hIL-5 mAb neutralization, mAb weretitrated against a constant concentration of rhIL-5. Twenty fivemicroliters of rhIL-5 (1 μg/ml final concentration) in RPMI completemedia were incubated with 25 μl of each anti-hIL-5 mAb, TRFK-5 (positivecontrol), and anti-OK11 mAb (negative control) (2-0.005 μg/ml 2 folddilutions) plus a media control for one hour at 37° C. Addition of BCL₁cells, labeling, and harvesting were the same as described above.

In comparison to the media control (no mAb), TRFK-5 served as a positivecontrol for neutralization. TRFK-5 was capable of neutralizingbiological activity of both rhIL-5 and rmIL-5. An irrelevant isotypematched mAb (murine IgG¹, kappa), anti-OKT11 had no significant effecton either rhIL-5 or rmIL-5. All murine mAb significantly neutralizedrhIL-5, with group B mAb having more neutralizing potential at 20 μg/mlthan group A mAb. None of these antibodies however had any significanteffect on rmIL-5. Neutralization by all mAb were shown to be dosedependent. FIG. 2 demonstrates this dependency for IL5.7 and IL5.15,representatives of group A and group B mAb respectively, and TRFK-5.Maximum neutralization of 1 μg/ml of rhIL-5 was achieved with 20 μg/mlof both mAb as well as TRFK-5. Neutralization diminished to zero for allmAb at 5 μg/ml. The isotype-matched control mAb, anti-OKT11, had noeffect of rhIL-5 activity over the entire range of MAb concentrations.

EXAMPLE 6 WESTERN BLOT ANALYSIS

To assess potential conformational requirements for epitope recognitionby each mAb, Western analyses of both reduced and nonreduced rhIL-5 wereconducted using each mAb as the primary detecting antibody.

Thus, recombinant hIL-5 was diluted to approximately 15 ng/lane andelectrophoresed through a 12% polyacrylamide SDS gel under eitherreducing or nonreducing conditions. For each gel a Rainbow molecularweight markers (Amersham, Arlington Height, Ill.) lane was run tomonitor electrophoresis and efficiency of transfer. Protein wastransferred to a PVDF immobilon-P membrane (Millipore, Bedford, Mass.)in a Trans-Blot Cell (BioRad, Hercules, Calif.) 500 mA for two hours at15° C. Membranes were blocked with PBS 0.05% tween 0.5% nonfat dry milkO.N. at 4° C. Membranes were then assembled in a Miniblotter 16(Immunetics, Cambridge, Mass.). Each mAb was diluted to 100 μg/ml inblocker and 150 μl added to the appropriate lane and incubated for onehour at room temperature. Membranes were developed using an EnhancedChemiluminescence (ECL) Western blotting kit and Hyperfilm-MP (Amersham,Arlington Height, Ill.). Between each step membranes were wash threetime with PBS 0.05% tween 0.5% BSA for 10 min on a shaker with a finalPBS 0.5% tween rinse.

All mAb were found to bind denatured nonreduced rhIL-5, however tovarying degrees (FIG. 3A). The mAb could be grouped based on intensityof the ECL signal (in decreasing order) IL5.4-7, IL5.1-3,10-11, IL5.8-9,and IL5.12-15. No mAb was bound denatured reduced rhIL-5.

Western analysis thus provides a means of further dividing the mAbgroupings. By qualifying the intensity of signal from a normalizedconcentration of mAb and a fixed amount of hIL-5, four levels ofrelative intensity were defined (FIG. 3A). All group B mAb demonstratedpoor binding by Western and thus could not be further subdivided.However, the group A mAb demonstrated three different degrees ofintensity and thus could be subdivided by this characteristic. Thesedifferences may be due to multiple epitopes or merely to affinitydifferences to recognition of different epitopes or merely differencesin affinity to the same epitope. What can be concluded is that allepitopes have some conformational dependency, since no mAb were found tobind reduced hIL-5, even at a levels as high as one microgram (data notshown). Therefore all mAb binding is dependent on at least one of thehIL-5 disulfide linkages. Finally ELISA combinations IL5.7:b-IL5.7(group A), IL5.15:b-IL5.15 (group B), and TRFK-5:b-TRFK-5 were allcapable of detecting hIL-5 (FIG. 4), demonstrating that each of thesethree classes of neutralizing epitopes must be present as aconformationally identical pair on the hIL-5 dimer.

Further analysis of other mAb combinations provides insight into therelative location of each epitope pair. The TRFK-5:b-TRFK-5 and theIL5.15:b-IL5.15 combinations were orders of magnitude less sensitivethan the IL5.7:b-IL5.7 sandwich ELISA. This suggests that both group Aepitopes are more available to simultaneous binding than those for agiven group B mAb or TRFK-5. The less sensitive group B or TRFK-5sandwich ELISA argues that having one of a given epitope bound by thecapture mAb limits the availability for binding of the detecting b-mAb.This general conclusion that the group B and TRFK-5 epitopes are incloser proximity than the group A epitopes is further supported by ELISAwhich utilized unlike pairs. The IL5.7:b-1L5.15 pair improvedsensitivity over the IL5.7:B-IL5.7 combination, suggesting that perhapseven partial restriction on the second group A epitope exists. Capturingwith a group A mAb leaves one group B domain virtually free to bind tothe respective detecting b-mAb. The fact that the IL5.7:b-TRFK-5sandwich was less sensitive than the IL5.7:b-IL5.15 suggests thatcapturing with a group A mAb limits the TRFK-5 epitope more than that ofgroup B. This further supports the conclusion that TRFK-5 and group Bepitopes must be different. The most sensitivity combination and thusthe combination suggesting the best detection epitope availability isthe IL5.15:b-IL5.7 combination. Capture with IL5.15 resulted in theavailability of nearly two other A epitopes, the most of anycombination.

In summary, utilizing sensitive mAb combinations, highly sensitive andspecific hIL-5 ELISA were developed. The sensitivity for theIL5.15:b-IL5.7 sandwich ELISA was 15 pg hIL-5/ml, which rivals otherreported ELISA (K. C. Allison, et al., J. Immunol. 146:4197 (1991);McNamee, L. A., et al., J. Immunol. Meth. 141:81 (1991); Fukuda, Y., etal., J. Immunol. Meth. 143-89 (1991)) and is capable detecting hIL-5 inbiological fluids. At the same time this assay proved to be veryspecific, as undiluted sera and concentrated BAL samples from normalcontrols generated no detectable signal.

EXAMPLE 7 DEVELOPMENT OF AN hIL-5 SANDWICH ELISA

In an attempt to develop a highly specific and sensitive hIL-5 ELISA,various combinations of capture mAb and detection b-mAb were employed(FIG. 4).

Thus, polystyrene ELISA plates (Corning, Corning, N.Y.) were coated with50 μl of a given capture mAb (5 μg/ml in PBS) and incubated overnight at4° C. Plates were blocked with PBS 0.5% tween 10% calf serum. Fiftymicroliters of standard dilutions of affinity purified rhIL-5 (intriplicate) and dilutions of blinded samples (in triplicate) wereincubated. Sera samples were run without further processing, whilebronchoalveolar (BAL) fluid was concentrated 20 times in an Amiconprotein concentrator using a 10 kD cut-off filter. Biotinylated mAb(1/500) were used for detection of bound hIL-5. The strepavidin-alkalinephosphatase/MUP system described above was used to develop the assay.Unless otherwise specified, all incubation were for one hour at roomtemperature and between incubations plates were washed five times withPBS 0.05% tween.

Serum was collected from a patient with eosinophilic pneumonia. BALfluid was collected from a patient with acute lung rejection. All othersamples were from normal individuals.

The previously described TFFK-5:b-TRFK-5 sandwich was again demonstratedto have a relatively poor sensitivity on the order of tens of ng/ml. Thecombination of IL5.15:b-IL5.15 (group B representative) proved just asinsensitive and had a lower signal throughout is usable range whencompared to the TRFK-5:b-TRFK-5 ELISA. The combination of IL5.7:b-IL5.7(group A representative) was slightly more sensitive approaching a lowerlimit of 1 ng/ml. Further enhancement of sensitivity was achieved usingIL5.7 as the capture antibody and detecting with b-IL5.15 (lower limitapproaching 100 pg/ml). However the best combination with the greatestsensitivity was achieved using the IL5.15:b-IL5.7 sandwich whose lowerdetection limit was 15 pg/ml (P<0.01). This sensitivity could not beimproved with a cocktail of all b-mAb. Sera from a patient witheosinophilic pneumonia and thirteen normal controls were assayed forhIL-5 (Table III). Although no hIL-5 was detected in any of his normalcontrol samples, hIL-5 was measurable in the serum of thehypereosinophilic patient (Table III). In addition, a 20-foldconcentration BAL from a patient undergoing lung transplant rejectioncontained measurable quantities of hIL-5, whereas hIL-5 was not detectedin similarly concentrated BAL from a healthy control individual (TableIII). In Table III, BAL fluid was concentrated 20 times; all sampleswere assayed in triplicate, and reported values are the mean ±SE.Control sera values represent th mean of 13 independent samples; a zeroindicates the absence of a signal over background.

                  TABLE III                                                       ______________________________________                                        Detection of hIL-5 in Biological Fluids                                              Sample       hIL-5 (pg/ml)                                             ______________________________________                                        Eosinophilic Serum                                                                            2083 ± 112                                                   Control Sera 0                                                                Inflammatory BAL 174 ± 9                                                   Control BAL 0                                                               ______________________________________                                    

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed is:
 1. A murine monoclonal antibody selected from thegroup consisting of IL5.1-IL5.15 and fragments thereof, wherein saidantibody or fragment:(A) specifically binds to human interleukin-5; (B)substantially fails to bind to murine interleukin-5; and (C) in thepresence of human interleukin-5 and a second murine monoclonal antibodyselected from the group consisting of IL5.1-IL5.15, forms a detectablemolecular complex that comprises said antibody, said interleukin-5, andsaid second murine monoclonal antibody.
 2. The murine monoclonalantibody according to claim 1, wherein the monoclonal antibody is IL5.1or a fragment thereof.
 3. The murine monoclonal antibody according toclaim 1, wherein the monoclonal antibody is IL5.2 or a fragment thereof.4. The murine monoclonal antibody according to claim 1, wherein themonoclonal antibody is IL5.3 or a fragment thereof.
 5. The murinemonoclonal antibody according to claim 1, wherein the monoclonalantibody is IL5.4 or a fragment thereof.
 6. The murine monoclonalantibody according to claim 1, wherein the monoclonal antibody is IL5.5or a fragment thereof.
 7. The murine monoclonal antibody according toclaim 1, wherein the monoclonal antibody is IL5.6 or a fragment thereof.8. The murine monoclonal antibody according to claim 1, wherein themonoclonal antibody is IL5.7 or a fragment thereof.
 9. The murinemonoclonal antibody according to claim 1, wherein the monoclonalantibody is IL5.8 or a fragment thereof.
 10. The murine monoclonalantibody according to claim 1, wherein the monoclonal antibody is IL5.9or a fragment thereof.
 11. The murine monoclonal antibody according toclaim 1, wherein the monoclonal antibody is IL5.10 or a fragmentthereof.
 12. The murine monoclonal antibody according to claim 1,wherein the monoclonal antibody is IL5.11 or a fragment thereof.
 13. Themurine monoclonal antibody according to claim 1, wherein the monoclonalantibody is IL5.12 or a fragment thereof.
 14. The murine monoclonalantibody according to claim 1, wherein the monoclonal antibody is IL5.13or a fragment thereof.
 15. The murine monoclonal antibody according toclaim 1, wherein the monoclonal antibody is IL5.14 or a fragmentthereof.
 16. The murine monoclonal antibody according to claim 1,wherein the monoclonal antibody is IL5.15 or a fragment thereof.
 17. Amurine hybridoma cell line that produces a monoclonal antibody selectedfrom the group consisting monoclonal antibodies IL5.1-IL5.15, whereinsaid monoclonal antibody:(A) specifically binds to human interleukin-5;(B) substantially fails to bind to murine interleukin-5; and (C) in thepresence of human interleukin-5 and a second murine monoclonal antibodyselected from the group consisting of IL5.1-IL5.15, forms a detectablemolecular complex that comprises said antibody, said interleukin-5, andsaid second murine monoclonal antibody.
 18. The murine hybridoma cellline according to claim 17, wherein the hybridoma produces monoclonalantibody IL5.1.
 19. The murine hybridoma cell line according to claim17, wherein the hybridoma produces monoclonal antibody IL5.2.
 20. Themurine hybridoma cell line according to claim 17, wherein the hybridomaproduces monoclonal antibody IL5.3.
 21. The murine hybridoma cell lineaccording to claim 17, wherein the hybridoma produces monoclonalantibody IL5.4.
 22. The murine hybridoma cell line according to claim17, wherein the hybridoma produces monoclonal antibody IL5.5.
 23. Themurine hybridoma cell line according to claim 17, wherein the hybridomaproduces monoclonal antibody IL5.6.
 24. The murine hybridoma cell lineaccording to claim 17, wherein the hybridoma produces monoclonalantibody IL5.7.
 25. The murine hybridoma cell line according to claim17, wherein the hybridoma produces monoclonal antibody IL5.8.
 26. Themurine hybridoma cell line according to claim 17, wherein the hybridomaproduces monoclonal antibody IL5.9.
 27. The murine hybridoma cell lineaccording to claim 17, wherein the hybridoma produces monoclonalantibody IL5.10.
 28. The murine hybridoma cell line according to claim17, wherein the hybridoma produces monoclonal antibody IL5.11.
 29. Themurine hybridoma cell line according to claim 17, wherein the hybridomaproduces monoclonal antibody IL5.12.
 30. The murine hybridoma cell lineaccording to claim 17, wherein the hybridoma produces monoclonalantibody IL5.13.
 31. The murine hybridoma cell line according to claim17, wherein the hybridoma produces monoclonal antibody IL5.14.
 32. Themurine hybridoma cell line according to claim 17, wherein the hybridomaproduces monoclonal antibody IL5.15.
 33. A method for the detection ofhuman IL-5 in a sample, comprising the steps:(1) contacting said samplewith a first monoclonal antibody selected from the group consisting ofIL5.1-IL5.15 and fragments thereof, wherein said antibody or fragment:specifically binds to human interleukin-5; substantially fails to bindto murine interleukin-5; and in the presence of human interleukin-5 anda second murine monoclonal antibody selected from the group consistingof IL5.1-IL5.15, forms a detectable molecular complex that comprisessaid first antibody, said interleukin-5, and said second murinemonoclonal antibody,under conditions that permit formation of a complexbetween the first monoclonal antibody and human IL-5; (2) contactingsaid complex with a second monoclonal antibody selected from the groupconsisting of IL5.1-IL5.15 and fragments thereof under conditionsinsufficient to permit formation of a complex comprising IL-5 and bothmonoclonal antibodies; and (3) determining a level of any complex formedcomprising IL-5 and both monoclonal antibodies; wherein the level of thecomplex containing both monoclonal antibodies and IL-5 is correlated tothe concentration of IL-5 in the sample.
 34. The method of claim 33,wherein the first monoclonal antibody is selected from the groupconsisting of IL5.15 and fragments of IL5.15, and the second monoclonalantibody is selected from the group consisting of IL5.7 and fragments ofIL5.7.
 35. The method of claim 34, wherein the concentration of IL-5 inthe sample is equal to or greater than 15 pg/mL.